SAD phasing using iodide ions in a high-throughput structuralgenomics environment

Jan Abendroth • Anna S. Gardberg • John I. Robinson •

Jeff S. Christensen • Bart L. Staker • Peter J. Myler •

Lance J. Stewart • Thomas E. Edwards

Received: 19 November 2010 / Accepted: 14 February 2011

� The Author(s) 2011. This article is published with open access at Springerlink.com

Abstract The Seattle Structural Genomics Center for

Infectious Disease (SSGCID) focuses on the structure

elucidation of potential drug targets from class A, B, and C

infectious disease organisms. Many SSGCID targets are

selected because they have homologs in other organisms

that are validated drug targets with known structures. Thus,

many SSGCID targets are expected to be solved by

molecular replacement (MR), and reflective of this, all

proteins are expressed in native form. However, many

community request targets do not have homologs with

known structures and not all internally selected targets

readily solve by MR, necessitating experimental phase

determination. We have adopted the use of iodide ion soaks

and single wavelength anomalous dispersion (SAD)

experiments as our primary method for de novo phasing.

This method uses existing native crystals and in house data

collection, resulting in rapid, low cost structure determi-

nation. Iodide ions are non-toxic and soluble at molar

concentrations, facilitating binding at numerous hydro-

phobic or positively charged sites. We have used this

technique across a wide range of crystallization conditions

with successful structure determination in 16 of 17 cases

within the first year of use (94% success rate). Here we

present a general overview of this method as well as several

examples including SAD phasing of proteins with novel

folds and the combined use of SAD and MR for targets with

weak MR solutions. These cases highlight the straightfor-

ward and powerful method of iodide ion SAD phasing in a

high-throughput structural genomics environment.

Keywords Experimental phasing � Iodide ions � SAD �Selenomethionine � Structural genomics � Structure

determination

Abbreviations

MAD Multi-wavelength anomalous dispersion

MR Molecular replacement

NIAID National Institute for Allergy and Infectious

Diseases

PDB Protein Data Bank

PSI Protein structure initiative

SAD Single wavelength anomalous dispersion

SSGCID Seattle Structural Genomics Center for

Infectious Disease

Electronic supplementary material The online version of thisarticle (doi:10.1007/s10969-011-9101-7) contains supplementarymaterial, which is available to authorized users.

J. Abendroth � A. S. Gardberg � J. I. Robinson �J. S. Christensen � B. L. Staker � L. J. Stewart �T. E. Edwards (&)

Emerald BioStructures, 7869 NE Day Road West,

Bainbridge Island, WA 98110, USA

e-mail: tedwards@embios.com

URL: http://www.ssgcid.org

P. J. Myler

Seattle Biomedical Research Institute, Seattle,

WA 98109, USA

P. J. Myler

Departments of Global Health and Medical Education

& Biomedical Informatics, University of Washington, Seattle,

WA 98195, USA

J. Abendroth � A. S. Gardberg � J. I. Robinson �J. S. Christensen � B. L. Staker � P. J. Myler �L. J. Stewart � T. E. Edwards

Seattle Structural Genomics Center for Infectious Disease,

Seattle, WA, USA

123

J Struct Funct Genomics

DOI 10.1007/s10969-011-9101-7

Introduction

The mission of the Seattle Structural Genomics Center for

Infectious Disease (SSGCID) is to provide a blueprint for

structure-guided drug design targeting NIAID class A–C

infectious disease organisms [1, 2]. To meet this goal, the

SSGCID plans to solve more than five hundred crystal

structures of potential drug targets from infectious disease

organisms over a 5 year period. The Center for Structural

Genomics of Infectious Diseases (CSGID) is a companion

center of SSGCID and maintains a similar mission [3]. For

SSGCID, targets are selected either through an internal

target selection process or are requested by members of the

scientific community external to the SSGCID consortium.

The majority of internally selected target have a homolog

that is a validated drug target with a known structure,

inferring that in principle most targets may be solved by

molecular replacement (MR). However, in practice, not all

targets solve by MR due to low sequence identity,

numerous sequence homology gaps, conformation changes,

etc. Furthermore, other targets are selected through statis-

tical analysis of sequence-based annotations (Cadag, E.

et al. unpublished) and numerous targets requested by the

scientific community do not have a homolog with a known

structure, and thus MR is not possible. Given the SSGCID

protein production pipeline that generates native protein

samples, we pursued strategies for obtaining de novo phase

information that utilize native crystals.

Dauter and co-workers described single wavelength

anomalous dispersion (SAD) phasing using iodide ions

[4, 5], a method that has been successfully employed by

others [6–11]. Native crystals are soaked into a solution

containing high concentrations of iodide ions, data is col-

lected in house at Cu Ka radiation where the anomalous

signal for iodide ions is large (Fig. 1), and the phases are

estimated using a SAD experiment [12]. This method is

simple, inexpensive, quick, effective, and was predicted to

be ‘‘particularly suitable for high-throughput crystallo-

graphic and structural genomics projects’’ [8]. Here, we

describe the application of iodide ion SAD phasing to a

number of SSGCID targets that required experimental

phase determination, resulting in sixteen new structures in

1 year. These structures are perhaps the largest collection

of iodide phased structures obtained by a single scientific

collaboration.

Materials and methods

Protein expression and purification

Detailed SSGCID protocols were (for example, see [7, 13,

14]) or will be published elsewhere. Here, we present a

general overview of target cloning, expression, and puri-

fication. SSGCID targets were cloned using ligation inde-

pendent cloning [15] from genomic DNA when available

or from codon engineered synthetic genes [16, 17]. The

most commonly used SSGCID expression vector

(pAVA0421) encodes an N-terminal histidine affinity tag

followed by the human rhinovirus 3C protease cleavage

Fig. 1 Anomalous scattering

factors for iodide and selenium

across the energy range used in

macromolecular

crystallography. The image was

generated using the University

of Washington X-ray

Anomalous Scattering Server

developed and maintained by

Ethan A. Merritt

(http://skuld.bmsc.

washington.edu/scatter/)

which is based on an earlier

publication [59]

J. Abendroth et al.

123

sequence (the entire tag is MAHHHHHHMGTLE-

AQTQGPGS-ORF), although other vectors were used as

well. All SSGCID targets were forward and reverse

sequence verified. Proteins were expressed in E. coli using

BL21 (DE3) R3 Rosetta cells and autoinduction media [18]

in a LEX bioreactor. The cells were pelleted, frozen at

-80�C, and purified at one of three different purification

groups, all of which used slightly variant purification pro-

tocols reflective of different equipment. Briefly, cells were

re-suspended in lysis buffer, sonicated, and clarified by

centrifugation. The proteins were purified initially by

immobilized metal affinity chromatography. At this point

in the purification protocol optional removal of the

expression affinity tag was done for about 60% of all tar-

gets. The protein sample was incubated with 3C protease

followed by a second nickel affinity column in which the

tagless protein of interest appeared in the flow through. All

protein samples used for structure determination described

here were purified by size exclusion chromatography

equilibrated in 20 mM HEPES pH 7.0, 300 mM NaCl,

2 mM DTT, and 5% glycerol. Fractions containing

pure protein were collected, pooled, concentrated to

*20–30 mg/ml, and stored at -80�C prior to crystalliza-

tion experiments.

Crystallization

Crystallization trials were set up using a rational sparse

matrix approach [19] which utilized the JCSG? and PACT

crystallization screens from Emerald BioSystems or

Molecular Dimensions. Sitting drop vapor diffusion crys-

tallization trials were set up at 16�C using 0.4 ll of protein

and 0.4 ll of precipitant against 80 ll of reservoir in

Compact Jr 96-well crystallization plates from Emerald

BioSystems. High value targets such as viral, eukaryotic,

fungal or community request targets were set up in addi-

tional crystallization trials such as the ProPlex screen from

Molecular Dimensions, the CSHT, Index, and Salt Rx

screens from Hampton Research or the Wizard Full (I/II)

and Wizard III/IV screens from Emerald BioSystems.

About 9% of targets that entered crystallization trials

yielded a data set with diffraction limits of 2.5 A resolution

or better straight out of the primary screen. In general,

targets that produced crystals that diffract to better than

*3.5 A resolution but did not yield data sets suitable for

structure determination were optimized using a 96-well

gradient optimization screen designed and produced using

the E-Wizard screen builder from Emerald BioSystems.

Many targets were screened and optimized using the

Microcapillary Protein Crystallization System (MPCS)

[20, 21] by Emerald BioSystems. SSGCID utilized

numerous salvage pathways such as in situ proteolysis [22]

and seeding techniques [23]. Full crystallization conditions

for each target solved by iodide ion SAD are shown in the

Supporting Information.

Iodide ion soaking

Typically, crystals were soaked for up to 5 min but occa-

sionally as long as 2 h into a solution similar to the pre-

cipitant solution, but which was supplemented with

0.2–1 M iodide ions (Table 1). In general, the cation used

for iodide ion soaks was aligned with the cations of the

crystallization solution, while the anion of the crystalliza-

tion solution was replaced with iodide. Full iodide ion

soaking conditions for each target solved by iodide ion

SAD are shown the Supporting Information.

Data collection and structure determination

Data sets were collected in house using either a Rigaku

007-HF or Rigaku SuperBright FR-E? X-ray generator

with Osmic VariMax HF optics and a Saturn 944 or Saturn

944? CCD detector. Diffraction images are available

through the CSGID web page (www.csgid.org). Data were

reduced with XDS/XSCALE [24] with the Bijvoet pairs

unmerged (i.e., Friedel setting at FALSE). Sites were

located using either phenix.hyss/phenix.autosol [25] or

SHELXD [26]. The anomalous substructure was refined

and extended, and phases were estimated using PHASER

EP [27] from the CCP4 suite [28] followed by density

improvement in PARROT [29]. Initial models were built

using automated building in BUCCANEER [30], followed

by model extension/rebuilding in ARP/wARP [31]. The

model was refined using SAD refinement with optimization

of the iodide ion occupancy in REFMAC [32]. Final

models were produced after numerous iterative rounds of

manual re-building in Coot [33] and refinement in REF-

MAC [32] using the merged data (i.e., Friedel setting at

TRUE in XSCALE [24]). The correctness of each structure

was examined, validated, and improved using Molprobity

[34, 35].

Results

SAD phasing using iodide ions

In essence, SAD phasing using iodide ions is comprised of

four steps. First, native crystals are soaked into a solution

similar to the precipitant reservoir supplemented with

iodide ions and cryoprotectant if necessary. Second, a data

set is collected in house at Cu Ka radiation where iodide

ions have an anomalous scattering coefficient of 6.9 e- (f00)(Fig. 1). Third, the iodide ion sites are located. Fourth,

SAD phasing using iodide ions

123

experimental phases are calculated using a SAD experi-

ment. We applied this method over a 1 year period to our

structural genomics structure determination pipeline,

resulting in the determination of 16 new structures from

seventeen targets, a 94% success rate (Tables 1, 2; Fig. 2).

Only five of the sixteen new structures followed a linear

path to successful structure determination, whereas the

other eleven required reiteration of one or more steps

before successful structure determination.

During the first step, we typically soak protein crystals

into a solution containing 1 M iodide ions, although in a

number of cases, the crystals were visibly damaged upon

soaking and/or diffracted poorly. Therefore, the soaking

step was repeated either at lower iodide ion concentrations

or in stepwise fashion across a range of iodide ion con-

centrations until a final concentration of 1 M was obtained.

For other targets we started with a more conservative

iodide concentration of 0.2 M which did not result in

successful structure determination (e.g., AnphA.00973.a),

whereas soaking at a higher concentration resulted in

successful structure determination. In one case (PDB ID

3LR5), the protein crystallized in a condition from the

PACT screen which contained 0.2 M NaI, and thus no

soaking was required.

In the data collection step, 360� of data were collected

for all data sets in order to maximize the multiplicity of the

data [8, 36], which should lower the error in the mea-

surement of Bijvoet pairs and thereby increase the accuracy

in the measurement of anomalous signal [37]. During this

step we discovered that the selection of the scaling reso-

lution had an influence in at least two cases for which

inclusion of all of the data did not lead to a successful

structure determination. For example, cutting the data at

lower resolution limits (2.5 A) led to the successful struc-

ture determination of a putative fructose-1,6-bisphosphate

aldolase from Coccidioides immitis (PDB ID 3PM6),

whereas the data could not be solved at 2.2 A resolution.

Others have reported similar results for phase calculation and

main chain building at 3 A, followed by refinement at 2.36 A

resolution [8]. The authors of the program SHELXD [26]

indicate that for anomalous-substructure searches truncation

of the data to 3.0–3.5 A resolution may be critical for some

sub-structure solutions [38]. For these data sets, we did not

observe any evidence for radiation damage, even for crystals

collected with long exposure times.

Unlike selenomethionine-labeled samples, the number of

iodide ion sites is not known a priori. Thus, locating the

heavy atom sites in the third step is an iterative process using

Table 1 Crystal structures determined by SSGCID using iodide ion soaks and SAD phasing

PDB ID targetDB IDa Protein nameb Phasing

resolution (A)cPrecipitant pH Iodide Soak time

3K9G BobuA.01478.a Plasmid partition protein 2.25 NaCl 6.5 1 M KI 2 9 1 h

3KM3 AnphA.00973.a Deoxycytidine triphosphate deaminase 2.1 PEG 3350 8.1 1 M KI 1 h, 15 min

3KW3 BaheA.00339.a Alanine racemase 2.95 PEG 3350 8.5 1 M KI 1 h

3LA9 BupsA.01663.a BpaA trimeric autotransporter adhesin 2.05 PEG 1500 4.0 1 M KI 1 h

3LR0 BupsA.00863.i risS periplasmic domain pH sensor at low pH 1.9 PEG 1000 4.2 1 M KI 1 h

3LR5 BupsA.00863.i risS periplasmic domain pH sensor at neutral pH 2.3 PEG 3350 6.5 0.2 M NaI Co-xtal

3LUZ BaheA.00759.a Extragenic suppressor protein suhB 2.05 PEG 400 7.5 0.75 M KI 1 h

3MD7 BrabA.11339.a b-lactamase like protein 2.0 PEG 3350 8.6 0.25 M KI 5 min

3MEN BupsA.10154.b Acetylpolyamine aminohydrolase 1.9 (NH4)2SO4 6.5 0.2 M NaI 1 h

3NJB MysmA.00358.i Enoyl-CoA hydratase 2.2 PEG 3350 8.5 0.4 M NaI 10 min

3O2E BaboA.10365.a Bol-A like protein 1.95 (NH4)2SO4 6.5 1.0 M NaI 4 9 5 min

3OIB MysmA.00247.b Acyl-CoA dehydrogenase 2.1 PEG 3350 8.5 0.5 M NaI 2 min

3OL3 MysmA.17112.a Ortholog of community request target Rv0543c 1.95 PEG 400 7.5 1 M NaI 4 min

3P96 MyavA.01155.a Phosphoserine phosphatase SerB 2.75 PEG 6000 6.0 1 M KI 4 min

3PFD MythA.00185.b Acyl-CoA dehydrogenase 2.1 PEG 8000 4.2 1 M NaI 20 min

3PM6 CoimA.00345.a Putative fructose-1,6-bisphosphate aldolase 2.5 PEG 8000 5.0 1 M NaI 15 min

a Organism identifiers correspond to the first four letters of the targetDB ID. Anph identifies Anaplasma phagocytophilum, Babo identifies

Babesia bovis, Bahe identifies Bartonella henselae, Bobu identifies Borrelia burgdoerferi, Brab identifies Brucella melitensis (biovar abortus),

Bups identifies Burkholderia pseudomallei, Coim identifies Coccidioides immitis, Myav identifies Mycobacterium avium, Mysm identifies

Mycobacterium smegmatis, and Myth identifies Mycobacterium thermoresistibileb BupsA.01663.a [7] and BupsA.00863.i were community request targets for which no homologous protein structure was available in the PDB;

neither of these targets contain internal methionine residuesc The resolution limits of the native data sets were 2.05 A for 3KW3, 1.27 A for 3MD7, and 2.05 A for 3P96

J. Abendroth et al.

123

one or more computational programs. We found this step to

be the most challenging for targets with a large asymmetric

unit because the programs compare the sites from multiple

solutions as a quality indicator. For example, acyl-CoA

dehydrogenase from Mycobacterium thermoresistibile

(PDB ID 3PFD) contained over one hundred iodide ions in

the final model (all of which had electron density above 5 r in

an anomalous difference Fourier map). Challenging the

programs to find 10 sites only returned 1 or 2 sites, because it

could not find the same 10 sites out of more than 100 total

sites. However, challenging the programs to find a higher

number of sites resulted in a successful structure determi-

nation. In contrast, crystals of the trimeric autotransporter

adhesin BpaA from Burkholderia pseudomallei were twin-

ned, and thus the number of sites was kept to a minimum to

avoid selecting strong sites from the minor twin fraction [7].

In that case, selecting six to ten sites yielded low quality

experimental electron density maps, whereas selecting only

two or four sites led to successful structure determination,

despite nine iodide ions in the final model. Based on the

number of amino acids per iodide ion for successfully

determined structures (Table 2), we recommend challenging

the programs to find one iodide ion per twenty amino acids of

the projected scattering mass.

The fourth step, SAD phase calculation, is also an iter-

ative process. After density improvement, the experimental

electron density maps were inspected for tertiary structure

such as a-helices and b-sheets as well as solvent channels

and correlation between iodide sites and the experimental

electron density. Any iodide sites which did not correlate

with the experimental electron density maps were elimi-

nated and the phases were re-calculated with only the real

iodide ion sites. This reiteration often improved the phase

quality and consequently resulted in more extensive auto-

mated structure building. For targets with a weak MR

solution from which a structure could not be determined

directly, the weak MR solution could be incorporated into

the SAD experiment. This combined use of SAD and MR

has been successful in four cases (Table 2 and see below).

In these cases, the increased number of sites indentified by

combined SAD/MR could be used in a subsequent SAD

experiment with no MR component to calculate the phases.

Success with a diverse set of proteins, crystallization

conditions, and crystal forms

The proteins used in these experiments derived from Gram

negative bacteria, fungi, and eukaryotes (Table 1).

Table 2 Crystallographic and phasing statistics for iodide SAD phased SSGCID structures

PDB ID Phasing

resolution (A)

Space group Multiplicitya SigAnob Residues

in AU

Solvent

content (%)

Iodide sitesc FOMd Method

3K9G 2.25 P43212 12.5 1.43 1 9 267 51 2/12 0.45 SAD

3KM3 2.1 H3 5.6 1.16 2 9 206 42 13/16 0.24 SAD

3KW3 2.95 C2 3.8 1.26 2 9 372 45 4/0 0.29 SAD/MR

3LA9 2.05 H3 5.4 2.08 1 9 178 29e 4/9 0.49 SAD

3LR0 1.9 P3221 11.2 2.33 1 9 123 46 6/6 0.44 SAD

3LR5 2.3 P212121 3.6 0.99 2 9 123 43 4/5 0.39 SAD

3LUZ 2.05 P21 3.8 1.22 2 9 267 37 6/13 0.50 SAD/MR

3MD7 2.00 C2221 6.5 1.34 2 9 272 40 11/0 0.49 SAD

3MEN 1.9 P212121 6.8 1.24 4 9 341 43 24/35 0.41 SAD

3NJB 2.2 I23 10.9 1.27 2 9 333 59 9/50 0.39 SAD

3O2E 1.95 P41212 10.3 1.70 1 9 90 25 5/9 0.53 SAD

3OIB 2.1 C2 3.7 1.78 2 9 403 48 15/50 0.55 SAD

3OL3 1.95 P212121 6.6 1.91 2 9 103 51 9/21 0.43 SAD

3P96 2.75 I222 7.7 1.70 1 9 418 53 15/0 0.41 SAD

3PFD 2.1 P21 3.7 1.19 4 9 389 48 8/109 0.53 SAD/MR

3PM6 2.5 P21 3.8 1.64 2 9 302 46 5/31 0.46 SAD/MR

a Overall multiplicity for anomalous scaled data; multiplicity for merged data is ca. twofold higherb SigAno is the mean anomalous difference in units of its estimated standard deviation (|F? - F-|/r) [24]c The number of iodide sites input into Phaser EP [27], followed by the number of iodide sites in the final model. For 3KW3, 3MD7 and 3P96

only the high-resolution native data was deposited into the PDB, and thus the final model contained no iodide ion sitesd Overall Figure of Merit (FOM) from Phaser EP [27] prior to density modificatione The solvent content was calculated based on the full length tagged protein. This protein only crystallized using in situ proteolysis with

chymotrypsin [22], and thus, the true solvent content is likely higher [7]

SAD phasing using iodide ions

123

Examination of the crystallization and soaking conditions

for the sixteen structures determined using iodide ion SAD

phasing revealed a number of different precipitants from

low to high molecular weight polyethylene glycols (PEG

400 to 8,000) and a variety of different salts including

ammonium sulfate and sodium chloride (Table 1 and

Supporting Information). These examples represent a broad

distribution of precipitants common to a rational sparse

matrix approach [19]. In addition, crystals grew and were

soaked over a wide pH range from 4.0 to 8.6 (Table 1),

spanning nearly the entire range of commonly used crys-

tallization screens. The space groups varied from low

symmetry (monoclinic, C2 and P21) to high symmetry

(cubic, I23) and from the most commonly observed space

group P212121 to rare space groups such as I23 (Table 2).

The number of residues in the asymmetric unit varied from

less than 100 to more than 1,500 (Table 2). The packing

density [39] ranged from a solvent content of 25%

(Vm = 1.64 A3/Da) to 59% (Vm = 3.04 A3/Da), spanning

the range commonly observed for protein crystals. The

phasing resolution ranged from 1.9 to 2.95 A with most

about 2.1 A; the native data sets for these structures ranged

from 1.27 to 2.3 A resolution (Table 1). Finally, the pro-

teins themselves are quite varied in structure and function

(Table 1; Fig. 2). For example, these structures contain

both novel (3LA9 [7]) and previously observed folds. The

Fig. 2 Crystal structures determined by SSGCID using iodide ion soaks and SAD phasing

J. Abendroth et al.

123

tertiary structure ranged from all a-helical (3OL3) to nearly

all b-sheet (3KM3). Different oligomeric states were

observed from monomeric to tetrameric. Apo and ligand

bound states were also observed. In all, these proteins,

crystallization conditions, crystal forms, and diffraction

properties are reflective of the SSGCID structure determi-

nation pipeline in general.

Types of iodide ion sites

The sixteen structures described here contained over 350

iodide ion sites combined, representing a wide variety of

iodide ion-protein interactions (Fig. 3). These sites can be

divided into several categories. As expected, we observed

binding of iodide ions near positively charged surface

residues, mostly arginine, lysine and to a lesser extent

histidine residues. Interestingly, we observed a number of

cases where the iodide ion displaced a surface exposed

negatively charged residue relative to the native structure,

in order to interact with a positively charged residue. A

second type of interaction we observed was the binding of

iodide ions to hydrophobic patches, such as binding prox-

imal to proline, methionine, or aromatic side-chains.

Again, in some cases, we observed displacement of a side

chain to an alternative rotamer conformation, relative to the

native structure, to accommodate the iodide ion in prox-

imity to a hydrophobic region. A third type of interaction,

which was one of the most frequently observed, involved

interactions with backbone amides via packing off the

O-C-N plane or binding to solvent exposed amide nitrogen

atoms. In the latter case, the iodide ion resided approxi-

mately 3.5–3.7 A away from the amide nitrogen atom. A

fourth type of interaction was the binding of iodide ions in

the vicinity of H-bond forming residues such as glutamine

and asparagine residues, and to a lesser extent threonine

and serine residues. Surprisingly, this category included

Fig. 3 Types of iodide ion binding sites. a Arginine-iodide ion

interaction network on the surface of MysmA.17112.a (PDB ID

3OL3), a putative uncharacterized protein from Mycobacteriumsmegmatis and an ortholog of community request protein My-

tuD.17112.a, Rv0543c from Mycobacterium tuberculosis (PDB ID

2KVC [60]) b Iodide ions binding along an a-helix of Co-

imA.00345.a, a putative fructose-1,6-bisphosphate aldolase from

Coccidioides immits (PDB ID 3PM6). Iodide ion IA forms a possible

anion–cation interaction with His21, while forming an interaction

with the side chain hydroxyl of Thr20 (3.3 A), an amide interaction

with Thr16 and a hydrophobic interaction with Phe17. Iodide ion IB

forms hydrophobic interactions with the side chains of Met298 and

Val14, while packing off the amide of Pro13-Val14. c Iodide ion

binding to the periplasmic domain of the risS pH sensor histidine

kinase from Burkholderia pseudomallei (BupsA.00863.i, a commu-

nity request target, PDB ID 3LR0). The iodide ion forms an

interaction with the backbone amide nitrogen of Asp122 (3.5 A) while

forming another interaction with the side chain of Ser119 (3.4 A) and

packing against two b-sheets. d Iodide ion binding off reduced flavin

adenine dinucleotide (FADH2) in the crystal structure of an acyl-CoA

dehydrogenase from M. thermoresistibile (MythA.00185.b, PDB ID

3PFD). An unbiased |Fo| - |Fc| map calculated from a model lacking

the cofactor is shown in green mesh contoured at 3.0 r. For each

panel iodide ions are shown as magenta spheres and an anomalous

difference Fourier map is shown in magenta mesh contoured at 5.0 r

SAD phasing using iodide ions

123

aspartic acid and glutamic acid residues for crystals

obtained at low pH (4.0–4.2), in which the carboxylic acid

side chains are expected to be protonated. Of course, many

iodide ions made multiple types of interactions with the

protein, such as residing next to the side chain of an

arginine residue while packing off an a-helix (Fig. 3).

Interestingly, we observed iodide ion binding off the uri-

dine-like ring of reduced riboflavin adenine dinucleotide

(FADH2) in the M. thermoresistibile acyl-CoA dehydro-

genase crystal structure solved at 2.1 A resolution (PDB ID

3PFD, Fig. 3d), an interaction which was observed in all

four protomers of the biological tetramer observed in the

asymmetric unit.

Case study 1: BolA-like protein from Babesia bovis

Babesia bovis is a tick-borne parasitic protozoan of the

phylum apicomplexa. It primarily infects cattle causing

babesiosis, a ‘malaria-like’ hemolytic anemia, although

occasionally infecting humans as well [40]. SSGCID target

BaboA.10365.a is a BolA-like protein from B. bovis.

Although the function is not fully understood, the

expression of a BolA-like protein in Escherichia coli was

reported to be linked to a change in morphology,

suggesting a function during cell division [41].

BaboA.10365.a is a small, 86-residue protein. Sequence

database searches revealed BolA-like proteins from Plas-

modium falciparum (51% sequence identify, PDB ID

2KDN; Buchko, G.W. et al. unpublished), from Mus

musculus (43% sequence identity, 1V9J [42]) and from

E. coli (34% sequence identity, 2HDM [43]) as closest

sequence homolog with a known structure. Unfortunately,

all of these solution NMR structures proved unsuitable for

MR [44]. BaboA.10365.a could be crystallized in a

tetragonal space group (P41212) with a small unit cell

(a = b = 66 A, c = 35 A) using ammonium sulphate as

the precipitant. The crystals lost order when the environ-

ment was changed too quickly, for example by introducing

iodide for phasing or ethylene glycol as cryoprotectant.

When the concentration of iodide and cryoprotectant were

simultaneously increased in several steps, well diffracting

crystals could be prepared. A highly redundant data set

with resolution limits of 1.95 A revealed strong anomalous

signal that extended to full resolution (SigAno 1.70,

anomalous correlation coefficient 59%). Four anomalous

sites were located in Phenix [25] and extended to nine

anomalous sites in PHASER EP [27]. After density mod-

ification with PARROT [29], ARP/wARP [31] could build

77 residues. While considering both enantiomorphic space

groups, the first model of BaboA.10365.a was obtained

literally minutes after data collection and data reduction

had finished. A post-hoc analysis of homologous structures

(Fig. 4) using the SSM/PDBeFOLD server [45] revealed

that sequence homologs have enough structural diversity,

rendering them unsuitable for MR. The closest structural

homolog is the BolA-like protein from M. musculus (1V9J,

[42]) with a RMSD of 1.9 A over 79 aligned residues. The

closest sequence homolog is the BolA-like protein from

P. falciparum (2KDN, Buchko, G.W. et al. unpublished),

which is less structurally similar: RMSD of 2.6 A over 76

aligned residues. The crystal structure of the BolA-like

protein from B. bovis described here (PDB ID 3O2E) is the

first crystal structure of a BolA-like protein, whereas all

other BolA-like protein structures have been solved by

solution NMR (Plasmodium falciparum PDB ID 2KDN,

Buchko, G.W. et al. unpublished; Mus musculus 1V9J

[42]; E. coli 2HDM [43]).

Case study 2: phosphoserine phosphatase SerB

from Mycobacterium avium

Orthologs from several Mycobacterium species (M. abscessus,

M. avium, M. bovis, M. leprae, M. marinum, M. paratuber-

culosis, M. smegmatis, M. ulcerans, and M. thermoresistibile)

are used in a salvage pathway to rescue M. tuberculosis

targets that fail at some stage of the SSGCID structure

determination pipeline. Phosphoserine phosphatase SerB

catalyzes the reaction of 3-phosphoserine to L-serine

(EC:3.1.3.3), the final step in the biosynthesis of ser-

ine. SerB from M. tuberculosis (targetDB MytuD.01155.a)

failed to produce diffraction quality crystals, and thus the

M. avium ortholog Ma SerB was entered into the SSGCID

pipeline. Ma Ser yielded a 2.05 A resolution native data set.

Fig. 4 Overlay of NMR solution structures of BolA-like proteins

from M. musculus (gray, PDB ID 1V9J [42]), P. falciparum(magenta, Buchko, G.W. et al. unpublished) and the crystal structure

of a BolA-like protein from B. bovis solved by iodide ion SAD

(green, PDB ID 3O2E). Iodide ions are shown as green spheres. For

simplicity only the ordered regions are shown

J. Abendroth et al.

123

The closest homologous structure in the PDB contained

44% sequence identity over less than half the Ma SerB

sequence with no homologous structure for the remainder of

the protein. Unsurprisingly, MR failed. Crystals used for

iodide phasing grew in 20% PEG 3350 and 0.2 M magne-

sium formate. For the first attempt, a crystal was soaked into

a mixture of 75% precipitant and 25% KI-saturated ethylene

glycol (final [KI] & 0.5 M), but did not yield sufficient

anomalous signal for structure solution (SigAno 1.34 for all

reflections to 3.15 A resolution). A second crystal was

soaked into 20% PEG 3350, 0.1 M magnesium formate,

1.0 M KI, and 25% ethylene glycol for 4 min, followed by

flash-cooling in liquid nitrogen and data collection, result-

ing in enhanced anomalous signal (SigAno 1.70). The

structure was solved at 2.75 A resolution using 15 iodide ion

sites located using phenix.autosol [25], followed by auto-

mated building and refinement using the high resolution

data set.

Like other SerB homologs, Ma SerB is a homodimer

with Mg2? and Cl- bound in the active site. By homology,

Mg2? is a required cofactor, and the chloride ion occupies

approximately the predicted phosphate position. Unusual

for SerB enzymes, Ma SerB consists of three domains

(Fig. 5), which is reflected in the low sequence homol-

ogy of Ma SerB with other SerB enzymes. The first

domain (residues 1–85) and second domain (residues

97–175) adopt the babbab ferredoxin fold, and there is a

domain swap between the two monomers of the dimer

via the long linker region between these two domains

(Fig. 2). The sequence identity between domains 1 and 2

is only 25%, but they superimpose with RMSD 1.4 A.

Domain 3 (residues 182–400) represents the conserved

core of the enzyme, and consists of a 6-stranded parallel

beta sheet, resembling a Rossman fold with a set of

extra helixes and anti-parallel beta strands inserted. Other

phosphoserine phosphatases overlap well on domain 3,

and some, such as that from Vibrio cholerae (PDB ID

3N28, Patskovsky, Y. et al. unpublished) match domain

2 poorly (Fig. 5). The N-terminal domain 1 of Ma SerB

appears to be unique.

Case study 3: putative fructose-1,6-bisphosphate

aldolase from Coccidioides immitis

Coccidioides immitis is a pathogenic fungus which causes

the potentially fatal systemic disease coccidioidomycosis

[46], also known as Valley Fever because it was thought

to originate in the San Joaquin Valley in California. Many

open reading frames from the genome of Coccidioides

immitis [47] are annotated as putative uncharacterized

protein, but contain sequence homology to proteins

of known function. One such case is the SSGCID target

CoimA.00345.a, which is annotated as a putative

uncharacterized protein. CoimA.00345.a shares 35%

sequence identity with 11% gaps over 70% of its

sequence with fructose-1,6-bisphosphate aldolase from

Giardia lamblia [48]. We obtained a 2.05 A resolution

native data set of CoimA.00345.a. Despite numerous

attempts, we were unable to solve this target via MR,

although we noted weak but plausible rotation (RFZ 6.0)

and translation scores (TFZ 3.8) in Phaser MR [27].

Soaking with 0.5 M NaI for 2 min yielded a 2.4 A res-

olution data set with weak anomalous signal (SigAno

0.91, 14% anomalous correlation) from which we were

unable to solve the structure using a SAD experiment.

Fig. 5 Sequence alignment and crystal structures of phosphoserine

phosphatase SerB from Vibrio cholera (bottom sequence, grayribbons, PDB ID 3N28, Patskovsky, Y. et al. unpublished) and

Mycobacterium avium solved by iodide ion SAD (top sequence, greenribbons, PDB ID 3P96). For simplicity, only one monomer of the

biological dimer is shown in each case. Domains 1, 2, and 3 of MaSerB correspond to residues 1–85, 97–175, and 182–400, respectively

SAD phasing using iodide ions

123

Another data set was obtained after soaking with 1 M NaI

for 15 min. Attempts to solve the structure at 2.2 A res-

olution failed, despite modest anomalous signal (SigAno

1.39, 52% anomalous correlation overall; 0.86, 7% in the

last shell). The resolution limits were trimmed to 2.5 A

resolution (SigAno 1.64, 62% anomalous correlation

overall; 0.96, 17% in the last shell), and despite locating

five sites, again we were unable to solve the structure.

Combining the weak MR solution and the anomalous sites

[49] in Phaser EP [27], we obtained a total of 31 heavy

atom sites (3 of which were incorrect), a FOM of 0.46,

and clearly interpretable experimental electron density

maps. Although the two fructose-1,6-bisphosphate aldol-

ases from the eukaryote G. lamblia and the fungi

C. immitis clearly share the same fold (Fig. 6), there are

numerous gaps and deletions in the protein sequence and

structure as well as considerable conformational hetero-

geneity, which results in an RMSD of 1.79 A for aligned

residues, a value which exceeds the common threshold for

success by MR [44].

Discussion

Experimental phasing in a structural genomics

environment

The most common method for de novo structure determi-

nation utilizes replacement of methionine residues with

selenomethionine and multiwavelength anomalous disper-

sion (MAD) or SAD experiments [50]. For success, this

method requires the completion of several steps. First, the

protein of interest must contain internal methionine resi-

dues or have them engineered into the protein sequence.

Second, a protein sample must be expressed using minimal

media supplemented with selenomethionine, a process that

often results in lower protein yields than native protein

expression. Third, the protein sample must be purified and

crystallized, a process that often requires re-optimization

relative to the native protein due to the increased lipo-

philicity of selenomethionine labeled proteins. Finally, this

method requires synchrotron radiation, preferably at a

tunable beamline, although several monochromatic beam-

lines such as ALS 5.0.1 and 5.0.3 have recently been

adjusted to an energy setting reflective of the selenium K

edge [51]. As a consequence of the numerous additional

steps required to solved structures by selenomethionine

SAD/MAD phasing, there is typically a lag time of several

months or more between obtaining native crystals that

diffract to high resolution and solving the structure.

One of the major goals of the Protein Structure Initiative

(PSI) was the elucidation of novel protein folds [52]. Thus,

for many PSI centers all of the protein samples destined

for crystallography were expressed as selenomethionine-

labeled proteins [53] with the intention of solving the

structures by traditional selenomethionine-based SAD or

MAD experiments. Although thousands of structures have

been solved using this method [50], there is additional cost

in reagents and time associated with solving structures via

this method in comparison with structures that may be

solved by MR. Given the PSI major goal of obtaining novel

protein folds, the additional cost is certainly justifiable.

Rather than investigate new folds, the goal of the SSGCID

is to determine structures of potential drug targets from

infectious disease organisms. Given that most protein

structures are expected to be solved by MR, all proteins are

expressed in native form in an effort to lower costs and

improve success rates; native proteins generally express at

higher levels and consequently crystallize at higher rates

than selenomethionine-labeled proteins. However, in

practice not all structures are solved by MR, and thus the

phases for these targets must be determined experimen-

tally. We could have adopted the selenomethionine-based

method; however, the SSGCID pipeline generates native

crystals, and thus, we decided to proactively explore

Fig. 6 Sequence alignment and crystal structures of fructose-1,6-

bisphosphate aldolases from Giardia lamblia (bottom sequence, grayribbons, PDB ID 2ISV [48]) and Coccidioides immitis solved by

combined iodide ion SAD and MR (top sequence, green ribbons,

PDB ID 3PM6). Iodide ions are shown as magenta spheres and the

catalytic zinc ions are shown as gray spheres. Anomalous difference

Fourier maps are shown in magenta mesh contoured at 5.0 r

J. Abendroth et al.

123

phasing options that use native crystals rather than waiting

months to obtain selenomethionine-labeled crystals.

There are a number of methods for heavy atom labeling

of native crystals and structure determination by isomor-

phous replacement and/or anomalous scattering methods

[54]. Methods developed within the past 10 years include

radiation induced phasing [55], covalent iodination [56],

non-covalent binding of an iodinated ‘‘magic triangle’’

[57], and bromine or iodide ion soaking and SAD or single

isomorphous replacement with anomalous scattering

(SIRAS) [4, 5]. For SSGCID, we wanted to use a method

that did not require toxic compounds such as mercury or

platinum, and which would be applicable over a wide range

of SSGCID targets. Therefore, we selected the use of

iodide ion soaking and SAD experiments [4, 5], which was

predicted to be particularly suitable for structural genomics

projects [8]. Over the past year, we applied this method to

seventeen structural genomics targets and determined six-

teen new structures. This method failed in only one case

(URE3-BP from Entamaeba histolytica, targetDB ID En-

hiA.01648.a, a community request target [58]), for which

MR, selenomethionine-based SAD/MAD, bromide ion

SAD/MAD, La3? SIRAS, K2PtCl4 SIRAS, HgCl2 SIRAS,

tungstate SAD, Cs? SAD, and sulfur SAD have also failed

thus far.

Why SAD phasing using iodide ions works

There are many reasons for the high success rate of

structure determination using iodide ion soaks and SAD

phasing by SSGCID. From the technical vantage point, the

modern in house X-ray generators, optics and detectors

used by SSGCID (see section ‘‘Materials and methods’’)

has had a significant impact on the success of this method.

Using previous generation in house X-ray equipment, for

many targets it would have taken 10 days to collect a full

360� data set required for high multiplicity, whereas the

current generation in house equipment (approaching 1011

Xrays/s mm2) rivals second generation synchrotrons in flux

and data collection times, ranging from 10 min to a few

hours. However, some synchrotron beamlines such as ALS

5.0.2 [51] have nearly as much flux at low energy where

the anomalous signal for iodide is high (e.g., 1.54 A) as at

higher energy (e.g., 1 A). Thus, this method is not exclu-

sive to in house data collection, although one must consider

the effects of radiation decay on the data collection

parameters [37]. In addition to improved hardware, current

software such as Phenix [25] and Phaser EP [27] has had a

significant impact on the success by making the identifi-

cation of anomalous sites rapid and accurate.

From the chemical vantage point, there are two major

reasons for the success of this method by SSGCID. First,

SSGCID targets are purified in moderately high salt con-

centrations (0.3 M NaCl), implying that SSGCID proteins

that crystallize were selected for stability in moderately

high salt concentrations. Thus, soaking into 0.2–1 M iodide

ions is unlikely to dramatically damage many of these

crystals. Second, at high concentrations, soft ions such as

iodides bind weakly to numerous sites on the surface of

proteins. These sites include binding to positively charged

residues, hydrophobic sites, amides, protonated residues,

etc. These types of interactions form regardless of crys-

tallization conditions including precipitant identity and pH,

or even dramatically different tertiary or quaternary protein

structure. These two features coupled with the high

anomalous signal of iodide at a wavelength of 1.5418 A

(f00 = 6.9 e- for iodide), which is higher than the theoretical

value of selenium at synchrotron radiation (f00 = 3.8 e- at

0.97946 A; white line effects at the selenium peak may

push f00 higher), are keys to the success of this method.

Conclusions

SAD phasing with iodide ions was applied to a diverse set

of structural genomics protein targets from bacterial,

fungal, and eukaryotic organisms, and over a wide range

of crystallization conditions common to most sparse

matrix approaches. The net result is rapid, effective, low

cost structure solution that addresses a bottleneck other-

wise created by the secondary preparation of selenome-

thionine-labeled crystals for a structural genomics

pipeline that normally generated native protein samples

and crystals. Although this method is not new, this is the

first time it has been used in a high-throughput structural

genomics environment. As a result, we obtained sixteen

new structures in 1 year, providing a wealth of informa-

tion with regard to the structural underpinnings of iodide

ion SAD phasing. Moreover, these cases demonstrate the

general applicability of this method for de novo structure

determination.

Acknowledgments This research was funded under Federal Con-

tract No. HHSN272200700057C from the National Institute of

Allergy and Infectious Diseases, National Institutes of Health,

Department of Health and Human Services. We wish to thank the

entire SSGCID team, especially members of the Crystal Core group,

Darren Begley, Doug Davies, and Robin Stacy for critical assessment

of the manuscript, and Banumathi Sankaran (Berkeley Center for

Structural Biology) for collection of a number of high resolution

native data sets.

Open Access This article is distributed under the terms of the

Creative Commons Attribution Noncommercial License which per-

mits any noncommercial use, distribution, and reproduction in any

medium, provided the original author(s) and source are credited.

SAD phasing using iodide ions

123

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